Nitric oxide (NO) is a radical messenger molecule with many functions, including neurotransmission, vasodilation, and pathogen killing. NO is produced by the family of nitric oxide synthase (NOS) isoforms. Activated macrophages contribute in inflammatory process. Upon various stimuli (cytokines, growth factors, bacterial, fungi, parasite and viral infection, inflammation), macrophages express iNOS (NOS2) which in turn produce gaseous signaling molecule, NO. NO derived from macrophages can kill a variety of pathogens (viruses, bacteria, parasites, tumor cells), but NO can also be toxic for cells producing NO. Thus, macrophages producing a high-output of NO need to protect themselves from its toxic effects. This grant application proposes to search for and characterize proteins expressed inside macrophages that regulate NOS2 activity and thus protect host cells against NO cytotoxicity. A genetic screen has identified a novel NOS2-associated Protein 110 kD (NAP110) that interacts with NOS2. Physical complexes between NOS2 and NAP110 were detected in a variety of experimental conditions in vitro and in vivo. Preliminary Data also show that NAP110 binds to NOS2 and inhibits NOS2 activity in transfected cells. The Specific Aims of this proposal are: (1) to analyze the domains of NOS2 and NAP110 that interact with each other; (2) to examine how NAP110 affects NOS2 function; (3) to localize NOS2 and NAP110 inside macrophages; (4) Does Chlamydia infection affect NOS2 and NAP110 localization? and (5) To examine the regulation NAP 110 gene at the transcription level. We will use various biochemical, molecular biology and cell biology techniques to study mechanisms of the interaction between NOS2 and NAP110 and their co-localization. They include; immunoprecipitation, immunoblotting, Northern blotting, two-hybrid yeast expression screen, in vitro translation and pull-down assay, transfection, genetic reporter assay and gel shift assay, and confocal immunofluorescense imaging analysis, differential and gradient centrifugation. This is a first report of a protein that interacts with NOS2 inside macrophages. The goal of these studies is to show how NOS2 and NAP110 interact with each other, and the consequences of this interaction. In particular, this application proposes to examine how NAP110 regulates NOS2 structure (homodimerization) and function (activity). These studies will increase our understanding of how NOS2 is regulated (negatively or positively) by NOS2-associated protein (s) and how macrophages and other cells that express NOS2 are protected against NO cytotoxicity.